SUMO, or Smt3 in Saccharomyces cerevisiae, is a ubiquitin-like protein that is post-translationally attached to multiple proteins in vivo. Many of these substrate modifications are cell cycle-regulated, and SUMO conjugation is essential for viability in most eukaryotes. However, only a limited number of SUMO-modified proteins have been definitively identified to date, and this has hampered study of the mechanisms by which SUMO ligation regulates specific cellular pathways. Here we use a combination of yeast two-hybrid screening, a high copy suppressor selection with a SUMO isopeptidase mutant, and tandem mass spectrometry to define a large set of proteins (>150) that can be modified by SUMO in budding yeast. These three approaches yielded overlapping sets of proteins with the most extensive set by far being those identified by mass spectrometry. The two-hybrid data also yielded a potential SUMO-binding motif. Functional categories of SUMO-modified proteins include SUMO conjugation system enzymes, chromatin-and gene silencingrelated factors, DNA repair and genome stability proteins, stress-related proteins, transcription factors, proteins involved in translation and RNA metabolism, and a variety of metabolic enzymes. The results point to a surprisingly broad array of cellular processes regulated by SUMO conjugation and provide a starting point for detailed studies of how SUMO ligation contributes to these different regulatory mechanisms.Many types of post-translational protein modifications alter protein function, and in some cases the modifying group is itself a protein. The prototypical example of this is ubiquitin, a small, highly conserved polypeptide that is reversibly linked to many different proteins (probably thousands) in the cell (1). Polypeptides distinct from but related to ubiquitin, called ubiquitinlike proteins or Ubls, can also be ligated to proteins (2, 3). Ligation to each Ubl has unique mechanistic and functional consequences. SUMO (Smt3 in the yeast Saccharomyces cerevisiae) is a divergent Ubl that has crucial roles in many organisms (4, 5). Vertebrates have four SUMO variants, SUMO1-SUMO4, whereas yeasts have only one. Only human SUMO1, and not the other SUMO variants, can substitute for the essential yeast protein (6).Activation and conjugation reactions involving SUMO have much in common with those of ubiquitin (reviewed in Refs. 2, 5, and 7). Attachment of substrates to Smt3/SUMO depends on a heterodimeric SUMO-activating enzyme (E1), 1 called Uba2-Aos1 in yeast, and a SUMO-conjugating enzyme (E2), Ubc9. Both enzymes form transient thiolester bonds with the C terminus of SUMO. Substrate recognition factors (E3s) that stimulate the transfer of SUMO from E2 to substrate have also been identified. SUMO, like ubiquitin, is always synthesized in precursor form, requiring enzymatic removal of a C-terminal peptide. Specialized proteases, called Ubl-specific proteases or Ulps, are responsible for these SUMO processing reactions and for reversing the post-translational attachment of SUMO to protein...

Data-dependent acquisition of MS/MS spectra from lipid precursors enables to emulate the simultaneous acquisition of an unlimited number of precursor and neutral loss scans in a single analysis. This approach takes full advantage of rich fragment patterns in tandem mass spectra of lipids and enables their profiling by complex (Boolean) scans, in which masses of several fragment ions are considered within a single logical framework. No separation of lipids is required, and the accuracy of identification and quantification is not compromised, compared to conventional precursor and neutral loss scanning.

Direct profiling of total lipid extracts on a hybrid LTQ Orbitrap mass spectrometer by high-resolution survey spectra clusters species of 11 major lipid classes into 7 groups, which are distinguished by their sum compositions and could be identified by accurately determined masses. Rapid acquisition of survey spectra was employed as a "top-down" screening tool that, together with the computational method of principal component analysis, revealed pronounced perturbations in the abundance of lipid precursors within the entire series of experiments. Altered lipid precursors were subsequently identified either by accurately determined masses or by in-depth MS/MS characterization that was performed on the same instrument. Hence, the sensitivity, throughput and robustness of lipidomics screens were improved without compromising the accuracy and specificity of molecular species identification. The top-down lipidomics strategy lends itself for high-throughput screens complementing ongoing functional genomics efforts.

Sterols and sphingolipids are considered mainly eukaryotic lipids even though both are present in some prokaryotes, with sphingolipids being more widespread than sterols. Both sterols and sphingolipids differ in their structural features in vertebrates, plants, and fungi. Interestingly, some invertebrates cannot synthesize sterols de novo and seem to have a reduced dependence on sterols. Sphingolipids and sterols are found in the plasma membrane, but we do not have a clear picture of their precise intracellular localization. Advances in lipidomics and subcellular fractionation should help to improve this situation. Genetic approaches have provided insights into the diversity of sterol and sphingolipid functions in eukaryotes providing evidence that these two lipid classes function together. Intermediates in sphingolipid biosynthesis and degradation are involved in signaling pathways, whereas sterol structures are converted to hormones. Both lipids have been implicated in regulating membrane trafficking.

In response to pheromone(s), Caenorhabditis elegans interrupts its reproductive life cycle and enters diapause as a stress-resistant dauer larva. This decision is governed by a complex system of neuronal and hormonal regulation. All the signals converge onto the nuclear hormone receptor DAF-12. A sterol-derived hormone, dafachronic acid (DA), supports reproductive development by binding to DAF-12 and inhibiting its dauer-promoting activity. Here, we identify a methyltransferase, STRM-1, that modulates DA levels and thus dauer formation. By modifying the substrates that are used for the synthesis of DA, STRM-1 can reduce the amount of hormone produced. Loss of STRM-1 function leads to elevated levels of DA and inefficient dauer formation. Sterol methylation was not previously recognized as a mechanism for regulating hormone activity. Moreover, the C-4 sterol nucleus methylation catalyzed by STRM-1 is unique to nematodes and thus could be a target for therapeutic strategies against parasitic nematode infections.

Novel photocaged lipids have been developed to release sphingosine at specific locations revealing a metabolic bias in ceramide synthesis based on subcellular location.

The worldwide increase in obesity and type 2 diabetes (T2D) represents a major health challenge. Chronically altered lipids induced by obesity further promote the development of T2D, and the accumulation of toxic lipid metabolites in serum and peripheral organs may contribute to the diabetic phenotype. Methods: To better understand the complex metabolic pattern of lean and obese T2D and non-T2D individuals, we analysed the lipid profile of human serum, skeletal muscle and visceral adipose tissue of two cohorts by systematic mass spectrometrybased lipid analysis. See editorial article: Sen P., Hyötyläinen T., and Orešič M. 2021. 1-Deoxyceramides-Key players in lipotoxicity and progression to type 2 diabetes? Acta Physiol (Oxf). e13635.2 of 20 | THOMAS HANNICH eT Al.

Nematode-specific iso-branched sphingoid base synthesis: drug-target against parasites and genetic model system for human diseases.